Three-dimensional printed calcium phosphate bone cement composite scaffolds for bone regeneration, precursor compositions, and methods of printing

ABSTRACT

Disclosed are 3D-printed scaffolds having high bone cement content, and in particular, high hydroxyapatite (HA) content. The disclosed methods and compositions provide the ability to print biocompatible scaffolds having patient-specific geometries with controlled porosity, microstructure, osteoconductivity, and mechanical strength. The scaffolds may be used for in vitro and in vivo craniofacial and dental applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of, and claims priority under 35 U.S.C. § 120 to, International Application No. PCT/US2021/019274, filed Feb. 23, 2021, and published under PCT Article 21(2) in English, which claims priority to U.S. patent application Ser. No. 16/894,128, filed Jun. 5, 2020, and to U.S. Provisional Patent Application No. 62/981,070, filed Feb. 25, 2020, the disclosures of which are incorporated herein by reference in their entireties.

BACKGROUND

A bone graft is a surgical procedure to repair, through tissue replacement and regeneration, bones or joints damaged by or the result of trauma, spinal fusion, tumor excision, and avascular necrosis. Bone grafts provide structural stability by replacing missing or damaged bone, for example, in the jawbone, bone grafts. Autografts and allografts are the two most common types of bone grafts. An autograft uses bone tissue that is transferred from one part of a patient's body to replace missing or damaged bone material in another part. Autografts are histocompatible and non-immunogenic, and offer all the imperative properties required of a bone graft material, such as osteoinduction, osteogenesis, and osteoconductivity. As such, autografts may be a preferred treatment. However, autografts, because of their source, are limited in supply, require extended operation procedures, and are related to donor site morbidity. A common alternative to autografts is allografts, which use bone tissue that is transplanted from one person to another. Allografts typically come from a living donor or a cadaver. Allografts are safe, ready to use, and can provide large amounts of bone material. In addition, allografts do not require additional surgical time for harvesting and recovery that may occur with an autograft. However, allografts do not have any cellular bone component because they are devitalized via irradiation or freeze-drying process leading to reduced osteoinductive properties. Finally, allografts are associated with risks of immunoreactions and transmission of infections.

Recent developments in bone tissue engineering have led to the use of artificial bone scaffolds as an adjunct to autografts, allografts, and other forms of bone grafts. These developments may incorporate additive biomanufacturing when fabricating biomimetic and complex organ structures. For example, synthetic scaffolds may be used to stimulate bone repair. Such scaffolds may be designed to be biocompatible and to exhibit porosity, mechanical properties, and osteoconductivity similar to those of native tissue. These scaffolds may have a specific form or geometry. Such scaffolds have been made through casting, mold, and electrospinning. More recently, scaffolds have been produced through three-dimensional (3D) printing, which has the advantage of providing a patient-specific geometry that may be derived, for example, from a computed tomography (CT) scan. Three-dimensional printing processes that include vat polymerization, powder bed fusion, material extrusion, and binder jetting, at low or high temperature, have been used for such bone substitute fabrication. Ceramic (or bioceramic) materials, including hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), α-tricalcium phosphate (α-TCP) combined with synthetic polymers, such as polycaprolactone (PCL), or metals (Titanium (Ti)), have been used.

However, current 3D printing processes for fabricating scaffolds suffer from significant deficiencies and thus are not able to produce high-resolution, biocompatible scaffolds. For example, current 3D printing methods are incapable of printing scaffolds with filament resolutions less than 200 Furthermore, with current 3D printing processes, the amount of ceramic material (such as hydroxyapatite) in the printing inks is less than about 30% of the total by weight (due to viscosity concerns associated with printing with the ink in printing environments), which results in printed scaffolds having low elasticity and low tensile and compressive strengths. Finally, current scaffolds are not printed at room temperature (RT) (i.e., between about 20-36° C.). Rather, current scaffolds typically are printed at much higher temperatures; temperatures high enough to prevent the viability of cells during the printing process. As a consequence, and as described herein, current 3D-printed scaffolds lack many bio-related advantages.

SUMMARY OF THE INVENTION

Disclosed are 3D-printed scaffolds having high bone cement content, and in particular, high hydroxyapatite (HA) content. The disclosed methods and compositions provide the ability to print biocompatible scaffolds having patient-specific geometries with controlled porosity, microstructure, osteoconductivity, and mechanical strength. The scaffolds may be used for in vitro and in vivo craniofacial and dental applications. Scaffolds having various shapes and sizes may be obtained by use of herein disclosed modifications in order to achieve desired mechanical properties required by differing applications. In an aspect, the scaffolds may be used for bone grafting and regeneration in humans.

The disclosed biocompatible 3D-printed scaffolds may be bioactive, osteoconductive, and biodegradable. These properties make the herein disclosed 3D-printed scaffolds useable for cell growth with greater efficacy and better predictability than is possible with current 3D scaffold printing methods. In an example, a 3D-printed scaffold may be printed along with living cells to generate cell-driven, functional tissue. Such a scaffold may be used in a human defect as a cell delivery mechanism. Furthermore, because of the fine resolution achievable with the herein disclosed compositions and methods, biomaterials may be deposited precisely in the scaffold to achieve a desired distribution, whether uniform or non-uniform. The 3D printing process may incorporate encapsulation of the biomaterials to prevent damage that otherwise might occur during scaffold material setting processes and from interactions of the biomaterials with components of slurries used in the printing process.

The herein disclosed scaffolds may be formulated from non-aqueous calcium phosphate cement (CPC) slurries used as 3D printing inks; in some embodiments the CPC slurries may include a hardening accelerator such as Na₂HPO₄. Three-dimensional printing in an aqueous bath helps avoid printer nozzle clogging resulting from possible rapid solvent evaporation at the nozzle tip. In some embodiments, a hardening accelerator such as Na₂HPO₄ may be added to the bath in an amount adjusted to control the hardening speed of the slurries. All 3D printing may be performed at room temperature (RT—as used herein a temperature between 20-36° C.), thereby allowing the use of a motor-driven syringe extruder with a room temperature working temperature rather than a hot-melting pneumatic extruder, with a working temperature in the range 60-350° C.

The slurry also includes a polymer dissolved in a non-aqueous solvent. The evaporation speed of polymer-solvents such as Tetrahydrofuran (THF) and Ethanol (EtOH) affect the slurry's rheological properties, which are strongly related to material printability. The viscosity of the CPC slurries may increase while the slurries load into the empty syringe; accordingly, slurry loading time may be minimized to avoid unwanted increases in slurry viscosity. Preferably, the non-aqueous solvent is miscible with the aqueous bath in which the 3D printing is carried out.

To print a scaffold according to a specific geometry and form for a specific application, the feed composition and movement of the injection nozzle relative to the printing substrate may be controlled by a computer executing a program of instructions.

In an example, a computer-controlled method for room temperature 3D printing a biocompatible, composition-controlled scaffold includes preparing a solid phase composition comprising a calcium phosphate cement powder; preparing a liquid phase composition comprising a dissolved polymer material; homogeneously mixing the solid phase composition and the liquid phase composition to create a homogeneous, bio-compatible slurry; disposing the slurry in a reservoir system coupled to a printing nozzle system, the printing nozzle system comprising at least one printing nozzle; submerging a printing substrate in a liquid bath disposed below the printing nozzle; under control of a computer, operating a motor to extrude the slurry, at room temperature, from the reservoir system through the printing nozzle system and to cause relative x, y, and z displacement between the printing nozzle system and the printing substrate, and to cause relative x, y, and z displacement between the printing nozzle system and each printing layer of the printing scaffold; employing a hardening accelerator to assist formation of the biocompatible, composition controlled scaffold; and maintaining the 3D printing scaffold fully submerged in the liquid bath during the entire 3D printing process.

In an example, a three-dimensional, biocompatible scaffold precursor composition for forming a bio-compatible polymer/bone cement composite scaffold includes a slurry, formed by mixing a solid phase of at least one calcium phosphate and calcium-containing compound with a non-aqueous liquid phase formed from dissolving a polymer in at least one non-aqueous solvent, where the polymer has either no or very low solubility in aqueous environments. In an aspect, the solid phase may be a mixture of tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and dicalcium phosphate anhydrous (DCPA; CaHPO₄). In an aspect, the liquid phase may include polyvinyl butyral (PVB) or polycaprolactone (PCL) in solution with a solvent such as Ethanol or Tetrahydrofuran. In an aspect, the solid phase comprises at least one calcium-containing compound selected from a group consisting of tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), α-tricalcium phosphate (α-TCP), β-tricalcium phosphate (β-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), calcium sulfate (CaSO₄), calcium sulfate hemihydrate (α- or β-CaSO₄.0.5H₂O), calcium carbonate (CaCO₃), calcium sulfate dihydrate (CaSO₄.2H₂O), calcium oxide (CaO) and calcium hydroxide (Ca(OH)₂).

In one aspect, the solid phase further comprises at least one water-soluble fluoride compound selected from the group consisting of an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, and a quaternary ammonium fluoride salt. In one aspect, the solid phase further comprises at least one water-soluble fluorosilicate or monofluorophosphate compound selected from the group consisting of an alkali metal fluorosilicate, an alkaline earth metal fluorosilicate, ammonium fluorosilicate, an alkali metal monofluorophosphate, ammonium monofluorophosphate, and other monofluorophosphates described in U.S. Pat. No. 4,046,872, herein incorporated by reference in its entirety, for example, magnesium monofluorophosphate and aluminum monofluorophosphate. In addition, the term “monofluorophosphate” includes water soluble monofluoropolyphosphates such as Na₄ P₃O₉F, K₄P₃O₉F, (NH₄)₄P₃O₉F, Na₃KP₃O₉F, (NH₄)₃NaP₃O₉F and Li₄P₃O₉F.

In an aspect, the liquid phase is formed from dissolving a polymer in at least one non-aqueous solvent, where the polymer has either no or very low solubility in aqueous environments. The polymer may be characterized as having a sufficiently low solubility to form a hardened mass in the presence of an aqueous solution.

In another example, a 3D printing precursor composition includes a slurry, as described herein, but with an added hardening accelerator. The slurry includes a solid phase formed from mixing, in an example, approximately 73% weight per weight (w/w) tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and approximately 27% w/w dicalcium phosphate anhydrous (DCPA; CaHPO₄), and a liquid phase formed from dissolving a polymer (that is insoluble or has low solubility in water) in a non-aqueous solvent. In other aspects of these examples, the solid phase may be composed of tetracalcium phosphate and dicalcium phosphate anhydrous having weight ratios less than approximately 97:3 and as low as approximately 8:92. In an aspect, the solvent may be Ethanol (EtOH) and Tetrahydrofuran (THF). In an aspect, the solid to liquid phases have a weight ratio of 0.75 to 1. In other aspects, the solid to liquid phase weight ratio may range from 0.1 to 1 up to 3 to 1, or from 0.5 to 1 up to 3 to 1. In yet another example, a 3D printing precursor composition includes a slurry, as described herein, and a hardening accelerator that is introduced during printing of the polymer/hydroxyapatite composite scaffold. In an example, the hardening accelerator is introduced into an aqueous or non-aqueous bath in which the scaffold is printed. In another example, the hardening accelerator is mixed with the slurry. In still another example, a 3D printing precursor composition includes a slurry, as described herein, and a hardening accelerator is provided in both the slurry and an aqueous or non-aqueous bath. In an aspect, the hardening accelerator may be disodium phosphate (Na₂HPO₄).

A three-dimensional, biocompatible scaffold precursor composition for room-temperature printing a bio-compatible polymer/hydroxyapatite composite scaffold includes a room-temperature slurry, comprising a mixture of a sold phase that includes a mixture of tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and dicalcium phosphate anhydrous (DCPA; CaHPO₄), and a liquid phase that includes a polymer in a solvent. The solvent may be Ethanol (EtOH) or Tetrahydrofuran (THF), and the polymer may be polyvinyl butyral (PVB), polycaprolactone (PCL), or poly lactic-co-glycolic acid (PLGA). A hardening accelerator may be added to the slurry during room-temperature printing of the polymer/hydroxyapatite composite scaffold. A three-dimensional, biocompatible precursor composition for room-temperature printing a 3D bio-compatible polymer/bone cement composite scaffold includes a room-temperature slurry formed as a mixture of a solid phase and a liquid phase. The solid phase is at least one calcium-containing compound, and may be one of tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), α-tricalcium phosphate (α-TCP), β-tricalcium phosphate (β-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), calcium sulfate (CaSO₄), Calcium sulfate hemihydrate (α- or β-CaSO₄.0.5H₂O), calcium carbonate (CaCO₃), calcium sulfate dihydrate (CaSO₄.2H₂O), calcium oxide (CaO) and calcium hydroxide (Ca(OH)₂).

Optionally, the solid phase further comprises at least one water-soluble fluoride compound selected from the group consisting of an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, and a quaternary ammonium fluoride. Optionally, the solid phase further comprises at least one water-soluble fluorosilicate or monofluorophosphate compound selected from the group consisting of an alkali metal fluorosilicate, an alkaline earth metal fluorosilicate, ammonium fluorosilicate, and a monofluorophosphate.

The liquid phase includes a polymer in a non-aqueous solvent, and the polymer is insoluble or has low solubility in water. A weight ratio of the solid phase to the liquid phase is between 0.1 to 1 and 3 to 1.

A computer-controlled method for room-temperature printing a composition-controlled product using 3D printing includes disposing a liquid reactant composition in a reservoir, the liquid reactant composition comprising a mixture of: a solid phase comprising a calcium phosphate cement (CPC) powder, and a liquid phase comprising a polymer material dissolved in a solvent, the polyvinyl material selected from a group consisting of polyvinyl butyral (PVB) and polycaprolactone (PCL), the solvent selected from a group consisting of Ethanol (EtOH) and Tetrahydrofuran (THF); at room temperature, extruding the liquid reactant composition by a computer controlling a motor-driven syringe extruder having an exit nozzle of diameter less than or equal to 210 micrometers; scanning, under control of the computer, a liquid reactant exit nozzle over a substrate while maintaining the substrate fully submerged in an aqueous bath; and depositing the liquid reactant composition onto the substrate, wherein the solvent evaporates to produce, under influence of a hardening accelerator, a biocompatible polymer/hydroxyapatite composite scaffold.

A computer-controlled method for room temperature 3D printing a biocompatible, composition-controlled scaffold includes preparing a solid phase composition comprising a calcium-containing cement powder; preparing a liquid phase composition comprising a polymer material dissolved in a solvent; homogeneously mixing the solid phase composition and the liquid phase composition to create a homogeneous, bio-compatible slurry; disposing the slurry in a reservoir system coupled to a printing nozzle system, the printing nozzle system comprising at least one printing nozzle; submerging a printing substrate in a liquid bath disposed below the printing nozzle; under control of a computer, operating a motor to extrude the slurry, at room temperature, from the reservoir system through the printing nozzle system and to cause relative x, y, and z displacement between the printing nozzle system and the printing substrate; employing a hardening accelerator to assist formation of the biocompatible, composition-controlled scaffold; and maintaining the 3D printing scaffold fully submerged in the liquid bath during an entire 3D printing process.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description refers to the following figures in which like numerals refer to like objects, and in which:

FIG. 1 is a schematic of a process for preparing polyvinyl butyral/calcium phosphate cement (PVB/CPC) slurries.

FIG. 2 illustrates the viscosity (ii) of different PVB/CPC slurries as a function of shear rate (1/s).

FIG. 3A comprises a schematic illustration of an example 3D printing system used to produce PVB/HA scaffolds in an aqueous bath.

FIG. 3B further illustrates the example 3D printing of the system of FIG. 3 .

FIG. 3C shows a cross-section of an example vessel and an aqueous bath shown in FIG. 3 .

FIG. 3D shows a schematic side view of an example 3D printing system component.

FIG. 3E shows a schematic side view of another example 3D printing system component.

FIG. 4 presents representative images of PVB/HA scaffolds printed using a 210 μm diameter nozzle according to the schematic process of FIG. 3B.

FIG. 5 presents a characterization of HA formation in a 3D printed scaffold.

FIGS. 6A-6B illustrate results of a scanning electron microscope (SEM) optical analysis of the PVB/HA scaffolds.

FIGS. 7A-7B present images of PVB/HVA scaffolds.

FIG. 7C presents calculated porosities of the PVB/HA scaffolds of FIGS. 7A and 7B.

FIG. 7D presents calculated surface areas of the PVB/HA scaffolds of FIGS. 7A and 7B.

FIGS. 8A-8D present characterizations of mechanical properties of the PVB/HA scaffolds under tension.

FIGS. 9A-9D present measurements of mechanical properties of the PVB/HA scaffolds under compression.

FIGS. 10A-10C illustrate results of osteoconductivity measurements for 3D printed scaffolds by staining.

FIGS. 11A-11F illustrate regulation of genes involved in osteoblast differentiation and mineralization.

FIGS. 12A-12B illustrate experimental setups for rheological measurements.

FIG. 13 illustrates osteoblast attachment and spread on a 3D printed scaffold based on immunostaining for F-actin (phalloidin) and DAPI (nuclei).

FIGS. 14A-14B illustrate the results of Live/Dead assays on 3D printed scaffolds.

DETAILED DESCRIPTION

The inventions disclosed herein relate to calcium phosphate cement (CPC) slurry compositions that are suitable for three-dimensional (3D) printing to produce bone cements, such as hydroxyapatite (HA), and a novel and nonobvious biofabrication method for 3D printing of biocompatible polymer/bone cement composite scaffolds such as polyvinyl butyral/hydroxyapatite (PVB/HA). In an example, the novel and nonobvious biofabrication methods disclosed herein are based on 3D printing of CPC slurries in an aqueous solution bath containing, in an aspect, sodium phosphate dibasic (Na₂HPO₄) as a hardening accelerator. In another example, the methods are based on 3D printing of a CPC slurry that further contains a hardening accelerator within the slurry in place of or in addition to a hardening accelerator in the aqueous solution bath. When the hardening accelerator is present only in the slurry, the aqueous solution bath may be water.

The inventors formulated example CPC slurries by mixing CPC powder (solid phase) and different types of polymer-dissolved solutions (liquid phase). In an example, the solid phase may be a mixture of tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and dicalcium phosphate anhydrous (DCPA; CaHPO₄). As a person skilled in the art would recognize, other suitable solid phase compounds include α-tricalcium phosphate (α-TCP), β-tricalcium phosphate (β-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), calcium sulfate (CaSO₄), calcium sulfate hemihydrate (α- or β-CaSO₄.0.5H₂O), calcium carbonate (CaCO₃), calcium sulfate dihydrate (CaSO₄.2H₂O), calcium oxide (CaO) and calcium hydroxide (Ca(OH)₂).

In some examples, the solid phase further comprises at least one water-soluble fluoride compound selected from the group consisting of an alkali metal fluoride (for example, potassium fluoride (KF), sodium fluoride (NaF), lithium fluoride (LiF), rubidium fluoride (RbF), caesium fluoride (CsF)), an alkaline earth metal fluoride (for example, calcium fluoride (CaF₂)), a silver-based fluoride (for example silver diamine fluoride or silver fluoride), stannous fluoride, ammonium fluoride, and a quaternary ammonium fluoride (for example, tetrabutyl ammonium fluoride). In other examples, the solid phase further comprises at least one water-soluble fluorosilicate or monofluorophosphate compound selected from the group consisting of an alkali metal fluorosilicate (for example, sodium fluorosilicate) and an alkaline earth metal fluorosilicate, ammonium fluorosilicate, and a monofluorophosphate (for example sodium monofluorophosphate, ammonium mono fluorophosphate).

In an example, the CPC powder was mixed approximately 73% w/w tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and approximately 27% w/w dicalcium phosphate anhydrous (DCPA; CaHPO₄). As used herein, “approximately” may refer to the capability to measure the solid phase compounds; alternately, approximately may refer to what is considered normal measurement practice in the art; alternately, approximately may refer to within 1 (one) percent (e.g., 72% to 74%). Similarly, “about” will have the same meaning as “approximately.” In other examples, other weight ratios were used. In an aspect, the liquid phase may include polyvinyl butyral (PVB) or polycaprolactone (PCL) in solution with a non-aqueous solvent such as Ethanol (EtOH) or Tetrahydrofuran (THF), or another water-miscible solvent (or solvent mixture). A person skilled in the art would recognize that other suitable polymers include poly lactic-co-glycolic acid (PLGA), Poly(ethylene glycol), Polyvinyl pyrrolidone (PVP), Poly(methyl methacrylate) (PMMA), Polyoxazoline, polyphosphoesters (PPE), or Dextran (and particularly, Dextran with high molecular weight greater between about MW=4,000,000 and MW=5,000,000). In general, the selected polymers have either no or very low solubility in aqueous environments. The polymer may be characterized as having a sufficiently low solubility if it can form a hardened mass in the presence of an aqueous solution.

Generally, 3D printing of such CPC slurries would be difficult because of (1) the high viscosity with a large amount of the CPC powder present in the slurry (with example solid to liquid phases having weight ratios between about 0.75 to 1 and 1 to 1), and (2) the rapid evaporation of the solvent. In an example, the inventors overcame these difficulties by 3D printing using a CPC slurry in an aqueous environment to form a solid HA structure, and further by accelerating HA hardening. In an example, the inventors used a motor-driven syringe extruder with small nozzles (e.g., diameters: 210 μm) to fabricate PVB/HA composite scaffolds, and all processes were carried out at room temperature. The PVB/HA composite scaffolds were successfully fabricated and tailored according to various periodic patterns regardless of which PVB solvent (EtOH or THF) was used for dissolving the PVB. The inventors tested the osteoconductivity of the PVB/HA composite scaffolds using Alkaline phosphatase (ALP), Alzarin Red (AR), and Von Kossa (VK). Cells cultured on EtOH75_HA scaffolds under mineralization conditions showed higher mineralization (˜2-fold) than cells cultured on THF75_HA scaffolds.

Furthermore, with current 3D printing processes, the amount of ceramic material (such as hydroxyapatite) in the printing inks is less than about 30% of the total by weight (due to viscosity concerns associated with printing with the ink in printing environments), which results in printed scaffolds having low elasticity and low tensile and compressive strengths. In contrast to current 3D scaffold printing methods and current printing inks, the inventors used precursor compositions having solid to liquid phases have weight ratios ranging from 0.1 to 1 up to 0.75 to 1; from 0.1 to 1 up to 3 to 1; from 0.5 to 1 up to 3 to 1; and from 0.75 to 1 up to 3 to 1. The inventors found that precursors compositions with these weight ratios, when used as disclosed herein, result in increased CPC loading in the printing ink, from a low of about 33% to 43%, and as high as 75%, which in turn results in much stronger scaffolds and scaffolds with higher resolution.

Three-dimensional printing using the herein disclosed printing methods and the CPC slurries and Na₂HPO₄ solution makes possible formation, in situ, of hydroxyapatite composite scaffolds at room temperature using syringe nozzle with a diameter of 210 μm or smaller. Advantages of room temperature (i.e., 20-36° C.) printing are disclosed herein. The methods and materials disclosed herein are compatible with many commercially available bioprinters commonly used in biofabrication and may be adapted to better replicate architectural and compositional requirements of individual tissues that are possible with traditional scaffold printing methods.

Recognizing that bone grafts made of a bioceramic material would be of importance for successful implantation and rapid osteointegration, and that additive manufacturing offers the ability to fabricate bone cement scaffolds, such as HA scaffolds, with defined macroporosity and improved mechanical properties, the inventors engineered 3D printed, in situ-formed, HA scaffolds using a rapid 3D printing procedure at room temperature. The inventors discovered that 3D printing of TTCP/DCPA in a Na₂HPO₄ bath results in the formation of HA in situ without the need for high temperature processes currently in use. The inventors' methods produced cell-integrated 3D printed scaffolds with controlled HA formation. In these methods, the inventors determined that material printability is related to many parameters, including particle size and size distribution, morphology and surface area of the powder, roughness and the ability of the powder to flow from a syringe extruder (flowability of the powders), and solubility/wettability/reactivity of the powder with the binder, such as PVB polymer. Several studies have shown that mean particle sizes of TCP particles in the range of 20-35 μm result in good 3D printing accuracy.

Although they have better flowability, larger particles tend to yield non-uniform layers of filaments leading to low resolution (filament size >200 μm) scaffolds. The inventors overcame this limitation by using smaller-sized (˜5 μm) TTCP particles, which stabilized powder bed homogeneity and yielded high-resolution 3D printed scaffolds. Additionally, the literature reports that the presence of solvent/polymer in HA slurries reduced the homogeneity of the slurries and, consequently, homogeneity of the printed scaffolds. To overcome this limitation, the inventors used a PVB-dissolved (25% w/v) solution-based slurries for printing. The example PVB/EtOH and PVB/THF solutions control the homogeneity of the slurries, thereby avoiding CPC particle separation and aggregation during 3D scaffold printing. The example PVB/EtOH and PVB/THF solutions without CPC showed Newtonian behavior, while at high shear rates (>100 1/s), the solutions turned into shear-thinning fluids. The presence of CPC in the example PVB/EtOH and PVB/THF solutions (i.e., the EtOH75 and THF75 slurries), changed this rheological profile, showing shear-thickening behavior at low shear rates (<0.25 1/s), while the behavior changed to shear-thinning behavior with increasing shear rates. Interestingly, the THF75 slurry showed slight fluctuations in the middle range of the shear rate, indicating some inhomogeneity within the slurry. This rheological profile (FIG. 2 ) of the EtOH75 and THF75 slurries further provides a controllable and consistent printing process that avoids inconsistencies and inhomogeneities.

The PVB polymer also contributes to the ultimate tensile strength (UTS) and the ultimate compressive strength (UCS) of the 3D printed scaffolds, which confirms the elastomeric properties of the scaffolds. The CPC reaction with a Na₂HPO₄ solution in the bath during printing produced HA in situ formation. Studies show that CPCs are promising for clinical applications due to their advantageous properties including bioactivity, osteoconductivity, injectability, and moldability. The solubility of TTCP and DCPA is at least one order of magnitude higher than that for HA at a pH range between about 6.5 and 9. This range is determined by the facts that DCPA is at least one order of magnitude more soluble the HA when the pH is about 6.5 or above, and that TTCP is at least one order of magnitude more soluble the HA when the pH is about 9 or below. At pH values higher than 9 and lower than 6.5, the solubility of the HA will approach that of either DCPA or TTCP. For the pH range between about 6.5 and 9, the solubility difference between HA on the one hand, and DCPA and/or TTCP on the other gives the ability to form HA in situ through a room temperature dissolution-precipitation reaction.

The ability to 3D print CPC could produce engineered scaffolds with designed mechanical properties and HA scaffolds for tissue regeneration applications, such as spinal and craniofacial injuries. For example, the herein disclosed EtOH75_HA and THF75_HA 3D printed 3D scaffolds exhibited differences in mechanical properties and scaffold shape. The EtOH75 slurry formed larger diameter filaments with large pores compared to THF75 slurry filaments, leading to a 40% larger scaffold surface area (FIG. 7D). The major reason for the differences between the EtOH75 and THF75 filaments is evaporation during the printing process. The THF vapor pressure (132 mm Hg) is ˜3 times higher than the EtOH vapor pressure (37 mm Hg), indicating that THF evaporates faster than the EtOH, leading to smaller pores in the scaffold. Although pore size is small in THF75 filaments, the overall percentage THF75_HA scaffold porosity is ˜1.5 times higher than that of the EtOH75_HA scaffold (FIG. 7C). Additionally, studies have demonstrated the effect of porosity on elastic modulus of the scaffolds. Similarly, the THF75_HA scaffold demonstrated ˜50% lower UTS and UCS, indicating that porosity may control the elastomeric properties of the scaffold. The porosity and the filament dimension of the scaffolds control the osteogenic and mineralization potential of the cells. Specifically, cells formed on EtOH75_HA scaffolds showed higher mineralization potential since they might spread and proliferate on the large filaments and between the large pores, resulting in a 2-fold increase in mineral deposition as shown by Von Kossa staining. Gene expression analysis of specific markers involved in osteoblast (OB) adhesion and bone matrix syntheses, such as OCN, COL1A2, and integrin β3 (ITGB3) showed higher expression on EtOH75_HA than THF75_HA scaffolds.

As described herein, a liquid phase of the two-phase slurry precursor mixture may be formed by dissolving a polymer in at least one non-aqueous solvent, where the polymer has either no or very low solubility in aqueous environments. The polymer may be characterized as having a sufficiently low solubility to form a hardened mass in the presence of an aqueous solution. Suitable polymers may be chosen according to their Hildebrand and/or Hansen solubility parameters, as is known to one skilled in the art. For example, the Hansen solubility parameters (HSP) have three components (δ_(D), δ_(P), δ_(H)) that set up a three-dimensional solubility space. Each polymer and solvent pair has its own set of HSP, i.e., (δ_(D), δ_(P), δ_(H)) that defines its location in the 3D solubility space.

The closer the points for a polymer and a solvent are within this 3D space, the more likely the compounds are to dissolve into each other. In the case of a polymer, only solvents within a certain range will dissolve the polymer. Polymers have an extra significant solubility number, called “Solubility Radius” or “Radius of Interaction” designated by “R.” The radius of interaction of a polymer with HSP (δ_(Dp), δ_(Pp), δ_(Hp)) defines a sphere in the solubility space, called the solubility sphere. The radius of the solubility sphere is calculated as (MPa)^(1/2). If the location of a given solvent/blend in the 3D solubility space falls within the solubility sphere of the polymer, that solvent/blend would be a good dissolving solvent/blend for that polymer. If the location of a given solvent/blend in the 3D solubility space falls outside the solubility sphere of the polymer, then the polymer would have no or low solubility in that solvent/blend. For example, for PVB, δ_(D)=18.6, δ_(P)=4.4, and δ_(H)=13, and for ethanol, δ_(D)=15.8, δ_(P)=8.8 and δ_(H)=19.4. The separation distance in the Hansen parameter space is about 9.6, and PVB dissolves in ethanol, since this distance falls within the solubility radius for PVB. However, water has δ_(D)=15.4, δ_(P)=16 and δ_(H)=42.3, and the distance is Hansen parameter space is about 32.1, which is greater than the solubility radius for PVB, thus PVB does not dissolve in water. For PCL, δ_(D)=17.5, δ_(P)=5 and δ_(H)=8.4, and THF has δ_(D)=16.8, δ_(P)=5.7 and δ_(H)=8. The distance in Hansen parameter space between PCL and THF is about 1.6, thus PCL dissolves in THF. PCL does not dissolve in water since the distance in Hansen parameter space (approximately 35.9) exceeds the solubility sphere radius for PCL.

In examples, polymers that may have high solubility with the solvent in the slurry and low (or no) solubility in the hardening accelerator bath or aqueous bath, and that may be used to produce the herein disclosed HA scaffolds include:

-   -   PVB dissolved in: acetic acid, acetone, methanol, Ethanol,         2-propanol, butanol, 2-butoxyethanol, cyclohexanone, benzyl         alcohol, 1-methoxy-propanol-2, butyl glycol, n-butyl, acetate,         ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide,         N,N-dimethylsulfoxide, NMP, and THF.     -   Polycaprolactone (PCL) dissolved in: THF, chloroform,         dichloromethane, carbon tetrachloride, benzene, toluene,         cyclohexanone and 2-nitropropane, acetone, 2-butanone, ethyl         acetate, dimethylformamide, and acetonitrile.     -   Poly Lactic-co-Glycolic acid (PLGA) dissolved in: THF, acetone,         ethyl acetate, and chlorinated solvents.     -   Poly-L-lactic acid (PLLA) dissolved in: chloroform,         dichloromethane (DCM).     -   Poly(ethylene glycol) (Solid PEG) dissolved in: acetone,         dichloromethane, ethanol (95%), methanol. (Liquid PEGs)         dissolved in: acetone, alcohols, benzene, glycerin, and glycols.     -   Polyvinyl pyrrolidone (PVP) dissolved in: methanol, Ethanol.     -   Polyacrylic acid (PAA) dissolved in: THF, methanol, Ethanol.     -   Poly(methyl methacrylate) (PMMA) dissolved in: Toluene,         Dichloromethane, Chloroform, and Acetone.     -   Polyoxazoline, polyphosphoesters (PPE): THF, acetonitrile,         chloroform, ethyl acetate (based on different type of PPE:         Poly((lactide-co-ethylene glycol)-co-ethyloxyphosphate)).     -   Dextran (and particularly, Dextran with high molecular weight         between about MW=4,000,000 and MW=5,000,000): methyl sulfide,         formamide, ethylene glycol, and glycerol.

The following table (Table 1) shows example CPC powders with molar ratios of TTCP to DCPA in a range from about 0.03 to 12 (with corresponding weight percentages of TTCP and DCPA between about 97:3% w/w and about 8:92% w/w, and molar Ca/P ratios between 1.95 and 1.06) that may be prepared in various examples for 3D-printing HA scaffolds. In examples, the molar Ca/P ratio is between 1 and 2, or between about 1.06 and 1.96, or more preferably between about 1.3 and 1.9.

TABLE 1 TTCP:DCPA TTCP/DCPA Molar Ca/P weight ratio (%) molar ratio ratio 97:3  12.1 1.96 96.5:3.5  10.0 1.95 93:7  5.00 1.91 92.5:7.5  4.55 1.90 84:16 2.00 1.80 80:20 1.50 1.75 73:27 1.00 1.67 64:36 0.67 1.57 57:43 0.50 1.50 52:48 0.40 1.44 47:53 0.33 1.40 43:57 0.29 1.36 40:60 0.25 1.33 20:80 0.095 1.16  8:92 0.032 1.06

In another example, using MCPA as a calcium-containing compound in the slurry, the Ca/P molar ratio may be as low as 0.5 to 1. In examples, other additives may be included in the solid phase of the slurry, including other calcium-containing compounds, and hardening accelerators, in powdered form or in non-aqueous solution. In examples, other additives such as a hardening accelerator may make up to about 10% of the weight of the solid phase of the slurry, or up to 5% of the weight of the solid phase of the slurry, or up to 1% of the weight of the solid phase of the slurry, or up to 0.5% of the weight of the solid phase of the slurry. In an example in which the hardening accelerator is powdered Na₂HPO₄, the hardening accelerator can make up between about 0.4% and 10%, or between about 0.45% and 9% of the weight of the solid phase.

The solid phase also may include a compound such as a carbonate (for example, NaHCO₃), a chloride (Cl) (for example, sodium chloride (NaCl), potassium chloride (KCl)), an alkali metal fluoride (for example, potassium fluoride (KF), sodium fluoride (NaF), lithium fluoride (LiF), rubidium fluoride (RbF), caesium fluoride (CsF)), an alkaline earth metal fluoride (for example, calcium fluoride (CaF₂)), a silver-based fluoride (for example silver diamine fluoride or silver fluoride), stannous fluoride, ammonium fluoride, a quaternary ammonium fluoride (for example, tetrabutyl ammonium fluoride), a fluorosilicate compound (for example, an alkali metal fluorosilicate such as sodium fluorosilicate, an alkaline earth metal fluorosilicate (such as magnesium fluorosilicate), and ammonium fluorosilicate), and a monofluorophosphate (for example sodium mono fluorophosphate, ammonium monofluorophosphate). These compounds may be added to the solid phase to achieve incorporation of CO₃, Na, Cl, and F ions into a printed bone cement formed by the herein disclosed 3D printing processes, while not inhibiting the formation of hydroxyapatite (or fluorapatite). Fluoride incorporation allows for the formation of a fluoride-substituted, or partially fluoride-substituted hydroxyapatite from the CPC reaction. When fluoride is added to CPC, it is readily incorporated into the apatite crystal structure of the printed bone cement, which can lead to a significantly improved crystallinity. Fluoride may also be incorporated into the printed bone cement as calcium fluoride. Fluoride may also act as a CPC setting accelerator. Fluoride incorporation into the apatite structure can also significantly lower in vivo resorption rates with increasing amount of fluoride incorporated into the apatite.

In examples, the herein disclosed CPC slurries may be formulated by mixing a CPC powder and a polymer solution in powder to liquid weight ratios in a range of about 0.1 to 1.0 CPC powder to liquid up to about 3:1 CPC powder to liquid, as shown in Table 2. Printability of the CPC slurries depends on the CPC powder to liquid ratio. The different CPC powder to liquid ratios will lead to either Newtonian or non-Newtonian behavior of the slurry thereby increasing or reducing (or creating non-uniformities), respectively, the filament printing resolution. In some embodiments, the CPC powder to liquid weight ratio is within a range of about 0.5 to 1.0 CPC powder to liquid up to about 3 to 1 CPC powder to liquid, or is within a range of about 0.75 to 1.0 CPC powder to liquid up to about 3 to 1 CPC powder to liquid.

TABLE 2 CPC Polymer CPC powder to powders solution liquid weight (gram) (gram) ratio 1 10 0.1:1 2 10 0.2:1 5 10 0.5:1 7.5 10 0.75:1  10 10   1:1 15 10 1.5:1 20 10   2:1 30 10   3:1

In an example, the CPC slurries (CPC powder+polymer solutions) may be printed in an aqueous environment to better form a bone cement such as HA and to improve the hardening process. Hardening time depends on the chemical composition and the concentration of the hardening accelerator(s), whether added to the aqueous solution, the slurry, or both the aqueous solution and the slurry. The hardening accelerators may have the following specifications: a) printed polymer in a CPC/polymer slurry is insoluble to the hardening solution, b) the accelerators may have pH (2-12) to form HA during the printing process and accelerate the hardening process, and c) the accelerators do not demonstrate toxicity. In an example, the hardening accelerator may be supplied with the aqueous solution in which the scaffold is printed. For example, the aqueous solution may contain sodium phosphate dibasic (Na₂HPO₄) as a hardening accelerator. In another example, the Na₂HPO₄ may be replaced by monosodium phosphate (NaH₂PO₄), trisodium phosphate (Na₃PO₄), ammonium phosphate (NH₄)₃PO₄), dipotassium phosphate (K₂HPO₄), sodium fluoride (NaF), potassium fluoride (KF), sodium acetate, potassium oxalate (C₂K₂O₄), sodium sulfate (Na₂SO₄), and sodium cacodylate (C₂H₆AsNaO₂). In addition, organic acids (e.g., glycolic, citric, tartaric, malonic, malic, maleic) may be used in the aqueous solution as a hardening accelerator. Finally, phosphate salts such as potassium and ammonium may be used as hardening accelerators.

In an example in which the herein disclosed scaffolds are printed using a pre-mixed composition including the CPC slurry (CPC powder+polymer solution), a hardening accelerator may be loaded into the syringe. The hardening times of the foregoing pre-mixture (CPC slurries+hardening accelerators) depend on the concentration of the hardening accelerators. The same hardening accelerators noted above, in powdered form or in non-aqueous solution form, may be included in the pre-mixed composition. Because the pre-mixed composition prints on the substrates quickly, the 3D printing process can be finished before the pre-mixed composition hardens in the syringe. In another example, a hardening accelerator may be loaded in a second syringe, and the scaffold may be printed using sequential printing of the CPC slurry from the first syringe and the hardening accelerator from the second syringe, causing a hardening reaction of the slurry in which the hardening accelerator also is deposited. In another example, a hardening accelerator also is contained within the aqueous environment in which the scaffold is to be printed.

In examples in which the solid phase includes TTCP and DCPA, the TTCP particle size varied in a range from 1 μm to 17 μm and with different combination of DCPA particle size in a range from 1 μm to 5 μm. The inventors note that slurries containing different particle sizes print scaffolds with different mechanical properties and HA formation. The inventors printed HA scaffolds for two particle size combinations: TTCP 17+DCPA 1 (larger syringe needle) and TTCP 5+DCPA 1 (smaller syringe needle). As explained herein, the particle size of DCPA should be smaller than that of TTCP or at least the same size, as shown in the Table 3 below. The choice of particle sizes used may promote faster hardening of the CPC material.

TABLE 3 TTCP (μm) DCPA (μm) 5-17 1-5 4 1-4 3 1-3 2 1, 2 1 1

For Table 3, the general rule is that the DCPA particle size should be smaller than or no greater than the TTCP particle size. This is because TTCP will dissolve faster than DCPA. If the TTCP particle is smaller than the DCPA particle, the majority of TTCP particles would completely dissolve before the DCPA particles dissolve. In this case, the 3D printed scaffold may have a less than optimum composition or a varying composition and as a result likely may be structurally weaker. If the particle sizes increase beyond the values in Table 3, then the hardening process for the product becomes slower, and a slower hardening process also may produce a weaker scaffold. Particle size also affects the homogeneity of the slurry. If the particle sizes are too large, the slurry may undergo phase separation, into essentially a solid phase and a polymer solution phase. Finally, as the particle sizes increase, nozzle clogging is more likely. Thus, the inventors determined that the ranges stated in Table 3 are the best ranges to provide the strongest structures with the shortest hardening times. Thus, for example, if TTCP is 4 μm, DCPA should be 1-4 μm.

In an example, the CPC slurries may be printed with nozzles (or needles) having diameters of 10, 30, 80, 100, 160 in addition to 210 achieving smaller (higher resolution) features within the scaffolds using smaller diameter nozzles. Larger diameter nozzles also may be used where high resolution scaffolds are not required.

Preparation of Calcium Phosphate Cement Powder (Solid Phase)

In an example, calcium phosphate cement (CPC) was formulated by mixing 73% w/w tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and 27% w/w of the dicalcium phosphate anhydrous (DCPA; CaHPO₄). To prepare TTCP, a mixture of DCPA (J. T. Baker Chemical Co.) and CaCO₃ (J. T. Baker Chemical Co.) was heated at 1500° C. for six hours in a bottom loading furnace (KEITH, EHSK-12, CA). Afterward, the mixture was quenched in air and then placed in a desiccator at room temperature for two days. Initially, the solid TTCP (which may be a chunk, or large and irregular particles) was dry ground in a planetary ball mill (Retsch PM4, Brinkman, N.Y.) to obtain a median particle size of 17 μm (TTCP17). The TTCP17 was further ground by a planetary ball mill for 24 hours to obtain a median particle size of 5 μm (TTCP5). Nozzle clogging was reduced significantly using the smaller size of TTCP (TTCP5). Finally, the DCPA was ground by a planetary ball mill for 24 hours to obtain a median particle size of 1 μm (DCPA1).

Preparation of PVB/CPC Composite Solutions (Liquid Phase)

In examples, two different types of Poly (vinyl butyral) (PVB, Mw˜60,000) (Scientific Polymer Inc., NY) solutions at a concentration of 25% w/v were prepared by (1) dissolving PVB in Ethanol (PVB/EtOH solution), and (2) dissolving PVB in Tetrahydrofuran (PVB/THF solution), each at 25° C. for 24 hours. Subsequently, CPC powder (TTCP5+DCPA1) was added to the PVB/EtOH or PVB/THF solution in a solid phase/liquid phase weight ratio of about 0.75 to 1, followed by magnetic stirring for 24 hours at 25° C. to create the PVB/EtOH/CPC slurry (EtOH75) and PVB/THF/CPC slurry (THF75), respectively. FIG. 1 illustrates this process schematically. To determine if the desired fabrication of 3D printed scaffolds was possible, and to ensure that a reproducible and fully-characterized biofabrication process could be defined, the inventors tested the printability of the two slurries. The viscosity of the slurries, EtOH75 and THF75, (see FIG. 2 , which illustrates the viscosity (ii) of different PVB/CPC slurries as a function of shear rate, with data expressed as the standard error of the mean (±SEM) for 4 measurements (N=4) by rheometer as shown in FIGS. 12A and 12B was measured. In the absence of CPC powder, the viscosity of the PVB/EtOH solution (1.094±0.009 Pa s) was 1.8 times greater than that of the PVB/THF solution (0.602±0.004 Pa s). The PVB solutions showed Newtonian flow behavior below 100 1/s. However, in the presence of a CPC powder, the viscosities of the PVB/EtOH/CPC (11.012±0.322 Pa s) and the PVB/THF75/CPC (8.970±0.698 Pa s) slurries were higher (approximately 10 times) than the PVB/EtOH solution and approximately 15 times higher than the PVB/THF solution, respectively. In addition, the CPC slurries exhibited shear thickening behavior at a low shear rate (<0.25 1/s), and then changed to shear thinning behavior over the remaining range of the shear rate.

Fabrication of PVB/HA Composite Scaffolds by 3D Printing

FIG. 3A is a schematic showing portions of an example 3D printing system 300 that the inventors used to produce Polyvinyl butyral (PVB)/Calcium Phosphate Cement (CPC) scaffolds, such as printed scaffold 380, in aqueous solution bath 340. Referring to FIG. 3A, a motor-driven syringe extruder 320 includes nozzle 310 (see expanded view) with nozzle opening 310 b having a nozzle diameter 310 a of 210 μm (27 gauge). Such a 3D bioprinter is available from Rokit Healthcare, INVIVO, South Korea. In an example 3D printing process, the syringe extruder 320 was filled with the EtOH75 slurry, printing parameters including printing speed, flow rate, and fill factor were entered into a computer system (not shown) used to control the 3D printing system 300, and a 3D printing process was executed. In another example, the THF75 slurry was used. In both studies, the printing process was carried out in a 0.1M sodium phosphate dibasic (Na₂HPO₄) aqueous solution bath 340 contained within vessel 330. The vessel 330 may be placed on a build plate or translation stage (not shown in FIG. 3A). The vessel 330 may be formed from glass or plastic or other material that allows adherence of the printing scaffold (that is, the scaffold being printed) to the vessel 330 during printing, while allowing removal of the printed scaffold from the vessel 330 after printing is complete. The vessel 330 may be a petri dish, a cell culture plate, a 24-well plate or the like. In some examples, vessel 330 is integrated with the translation stage of the 3D printing system 300.

FIG. 3B illustrates the example composition-controlled 3D printing system 300 in more detail. The system 300 includes processor sub-system 301, liquid flow control sub-system 302, and aqueous bath sub-system 303. The sub-systems 301-303 cooperate to produce a 3D, composition-controlled product such as biocompatible polymer/hydroxyapatite composite printed scaffold 380 (shown in FIG. 3A), which then may be used for bone repair. The processor sub-system 301 includes computer 372, which may execute machine instructions 374 to control motors that in turn control movement of other components of the system 300. In FIG. 3B, motor 376 is illustrated, and operates to move translation stage 370 in the xy plane during the printing process. Other motors (not shown) operate components of liquid flow control sub-system 302 such as the syringe extruder 320, which may be moved in the +/−z-directions as well as in the xy plane, and the extruder portion of which may be driven in the minus z-direction to force the slurry from the syringe extruder 320.

The system 300 also may include a liquid composition sub-system (not shown) having one or more mixing and storage components that are used to prepare and store the slurries, as well as other compositions that may be used to construct the 3D printed scaffold 380. In FIG. 3B, the syringe extruder 320 of the flow control sub-system 302 is shown to contain a single reservoir 322, which contains the desired slurry that is to be printed. The volume of the reservoir 322 may be 3 mL or 10 mL or other volume to allow printing of a structure. The vessel 330 contains an aqueous solution that, in an example, includes a hardening accelerator. In an example, the aqueous solution includes Na₂HPO₄ as the hardening accelerator. In an aspect, the Na₂HPO₄ may be replaced, in the aqueous solution bath 340, by various alternatives, including monosodium phosphate (NaH₂PO₄), trisodium phosphate (Na₃PO₄), ammonium phosphate (NH₄)₃PO₄), dipotassium phosphate (K₂HPO₄), sodium fluoride (NaF), potassium fluoride (KF), sodium acetate, potassium oxalate (C₂K₂O₄), sodium sulfate (Na₂SO₄), and sodium cacodylate (C₂H₆AsNaO₂). In addition, organic acids (glycolic, citric, tartaric, malonic, malic, maleic) may be used as a hardening accelerator. Finally, phosphate salts such as potassium and ammonium may be used as hardening accelerators. In examples where the slurry also includes a hardening accelerator, the aqueous solution bath 340 may be water. Upon printing, the aqueous Na₂HPO₄-containing bath liquid diffuses into the printed ink, causing the polymer to solidify and the CPC to harden by forming HA, resulting in in-situ polymer/HA formation and the fabrication of the desired composite scaffold. During this process, the solvents diffuse away from the printed ink into the bath liquid and/or evaporate.

The syringe extruder 320 is operated to control flow of reactant compositions from reservoir 322 into discharge component 324 and the rate of deposition of the slurry through nozzle 310. The nozzle 310 deposits the slurry onto a base of the vessel 330. In examples, the base is covered by the aqueous solution bath 340. In the configuration of FIG. 3B, vessel 330 is placed on the translation stage 370. In an aspect, rather than or in addition to controlling the translation stage 370 in the x-y plane, the computer 372 controls three-dimensional motion of the nozzle 310. Finally, FIG. 3B shows a scaffold 380 c, after printing is complete, but before removal from vessel 330.

In an example, the syringe extruder 320, discharge component 324, and nozzle 310 may be replaced by a multiple syringe extruders/multiple nozzle structures, with each syringe/nozzle structure having its own reservoir. With this multiple syringe/nozzle structure, the 3D-printed scaffold may be printed with slurries having differing compositions. In addition, the structure may permit deposition of cell-material on specific portions of the printing scaffold so as to achieve a desired non-uniform cell distribution. Finally, the structure may permit deposition of the slurry from one reservoir and deposition of a hardening accelerator from a second reservoir.

FIG. 3C shows a cross-section of the example vessel 330 and aqueous solution bath 340 shown in FIG. 3A. As shown in FIG. 3C, vessel 330 has sidewalls 332 to confine the aqueous solution bath 340 in the xy plane, and a base 334 that has a lower face 336 placed on and in direct contact with the translation stage 370 of the 3D printer system 300 (see FIG. 3B), and an upper face 338. Upper face 338 of base 334 of vessel 330 provides a build surface upon which a first printed layer of the 3D scaffold is printed directly. The aqueous solution bath 340 has a surface 340 a. In the examples described, printing was carried out at a constant speed of 5 mm/s (motion of the syringe extruder 320 (see FIG. 3A) in the xy plane, with respect to the upper face 338 of base 334). Other printing speeds also may be used. The printing speed, along with other printing parameters, including the nozzle (needle) opening size and the printing dimension or resolution parameters, determines the flow rate which the slurry should be dispensed from the nozzle 310 (not shown in FIG. 3C), in order to maintain the amount of slurry deposited per unit area at a (desired) constant value. The flow rate of the slurry is affected by slurry viscosity and by particle sizes within the slurry. The flow rate may be adjusted under computer control, for example by adjusting mechanically the pressure applied to the syringe extruder 320 of the 3D printer system 300.

In the experiments described herein, the syringe extruder 320 and the aqueous solution bath 340 were maintained at room temperature. In an example, as shown in FIG. 3D, the depth of the aqueous solution bath 340 (defined by surface 340 a of bath 340) was chosen to completely submerge the scaffold to be printed (380 a). That is, the level of the aqueous solution bath 340 was maintained higher than the planned height (in the z-direction) of the final, or completed, printed scaffold 380 (shown in FIG. 3A). Thus, the opening 310 b of nozzle 310 is below surface 340 a of bath 340 throughout printing of the completed, printed scaffold 380.

In other examples, the level of the aqueous solution bath 340 is maintained either at the same level or at a higher level than a top-most printing layer 380 b for the printing scaffold 380 a, as shown in FIG. 3E. In this example, surface level 340 b of aqueous solution bath 340 lies above the top-most printing layer 380 b of the printing scaffold 380 a, shown in FIG. 3D (i.e., above whichever layer of the printing scaffold 380 a that has been printed most recently). In order that the next layer-to-be-printed for the printing scaffold 380 a is printed within the aqueous solution bath 340, surface level 340 b of aqueous solution bath 340 is either at the same height as opening 310 b at the bottom of nozzle 310, or just submerges opening 310 b of nozzle 310. This allows the syringe extruder 320 to deliver another layer of material to deposit onto the top-most printing layer 380 b of the printing scaffold 380 a, increasing the height in the z-direction of the printing scaffold 380 a, and the deposited layer being formed within the aqueous solution bath 340. The initial, or, first layer of the printing scaffold 380 a is printed in contact with the base 334 of the vessel 330. A second layer of the printing scaffold 380 a then is formed on top of and in connection with the first layer. Subsequent layers are formed on top of, adjacent to, and in connection with a preceding underlying structural layer of the scaffold structure. After one layer of a structure is printed, the distance between the nozzle opening 310 b and the upper face 338 of the base 334 is increased, under computer control, in order to allow a subsequent layer to be printed on top of and in connection with the previously-printed layer. As the distance in the z-direction between the nozzle opening 310 b and the upper face 338 increases, additional aqueous solution can be added, under computer (or manual) control to the aqueous solution bath 340 using dispenser 390 that contains a solution of the same composition and molarity as that of the aqueous solution bath 340. The volume of aqueous solution to add can be determined by the change in height of the nozzle 310 in the z-direction, to ensure the top of the scaffold layer-to-be printed is either level with the surface level 340 b of the aqueous solution bath 340 or is submerged under the surface level 340 b of the aqueous solution bath 340.

Once a scaffold layer has been printed, in some examples, a second extrusion nozzle may be used, under computer control, to print biocells onto the printing scaffold 380 a. This may be achieved either within the aqueous solution bath 340, or at the top surface of the printing scaffold 380 a, at the interface between the aqueous solution bath 340 and the ambient environment (air) above, for example, by raising the second extruder nozzle in the z-direction such that it is above the surface of the aqueous solution bath 340.

During printing of a scaffold, the polymer initially hardens as it is deposited, since the polymer has no or low solubility in the aqueous solution bath 340. This solidification helps to provide the initial strength of the printing scaffold 380 a, before the co-deposited CPC materials from the slurry react with the aqueous solution to form a cement and harden.

In other examples, the scaffold-to-be printed also may be printed in an ambient (air) environment. An aqueous solution then may be sprayed onto the slurry material as it is deposited in order to solidify the polymer and/or to initiate cement formation and subsequent hardening of the CPC materials.

The scaffold may take any particular shape desired by a user. For example, the shape for the scaffold may be derived from an X-ray image, a computed tomography (CT) scan, a magnetic resonance imaging (MM) scan, or the like. Data from such a scan may be used to create a computer-aided design (CAD) model of the scaffold structure to be printed. CAD modeling may be used to produce any other suitable structure.

In the examples described, other printing parameter settings used for operation of the 3D printer were: filament gap (the distance between the exit of the needle or nozzle and the surface to be deposited on) of 210 μm, a layer thickness of 120 μm, and a lay-down pattern of 0°/90° (that is, adjacent layers are printed with orthogonal print directions as shown in the inset of FIG. 3A (schematic PVB/HA scaffold)). However, other values for filament gap and layer thickness may also be used to achieve different structural resolutions, and different lay-down patterns may also be used, determined by computer-controlled parameters for the 3D printer. In the above-described examples, with respect to the schematic system shown in FIG. 3B, the diameter of nozzle 310 is 210 μm. However, nozzles with other diameters may also be used, with nozzle diameters ranging between about 10 μm and 2100 μm. Three-dimensional printing parameters and their values used in the printing processes of the non-limiting examples described are summarized in Table 4. Parameters such as the needle diameter and needle length are physical parameters associated with the motor-driven syringe extruder 320. Values for other parameters, including print speed, fill density, input flow, layer height and temperatures are determined by the user, using computer software of the 3D printer.

TABLE 4 EtOH75 THF75 Needle (or nozzle) 210 210 diameter (um) Needle length (mm) 6.35 6.35 Printing speed (mm/s) 5 5 Fill Density (%) 50 50 Input flow (%) 250 250 Syringe temp (° C.) 25 25 Build plate temp (° C.) 25 25 Lay-down pattern (°) 0/90 Layer height (μm) 110-130 110-130

In examples, using a 3D bioprinter (Rokit Healthcare, INVIVO, South Korea), printing parameters may be chosen according to the ranges shown in Table 5. Fill density may be used to adjust the porosity of the structure by defining within a structure, regions where material is deposited and regions where no material is deposited. Input flow may be used to control the rate at which material is dispensed from the nozzle, and may be used to change the width and/or thickness of a deposited material. The minimum layer height that may be accurately produced is limited by the motor resolution in the z-direction of the 3D printer, and layer heights less than the filament gap and less than the nozzle diameter may be formed. In an aspect, printing is accomplished at room temperature, with syringe and plate (in the example schematic of FIG. 3C, the temperature of vessel base 334) temperatures of 25° C. However, in some examples where cells or bioinks are not required to be printed, temperatures up to about 80° C. for the syringe and/or the build-plate (translation stage 370 of FIG. 3B) may be used.

TABLE 5 Component Parameter Parameter Value Needle diameter (μm) 10-2100 Needle length (mm) 5-25  Printing speed (mm/s) 1-100 Fill Density (%) 1-100 Input flow (%) 0 < flow < 500 Syringe temp (° C.) 1-80  Build plate temp (° C.) 1-80  Layer thickness (μm) 10-2100

Turning to the experimental processes and results, hydroxyapatite (HA) formed upon reaction of the CPC component of the PVB/solvents/CPC slurries (EtOH75 or THF75) with Na₂HPO₄, which may be provided as a hardening accelerator in an aqueous bath. Afterward, the final 3D printed scaffolds, PVB/solvent/HA (EtOH75_HA or THF75_HA), were dried at room temperature for 48 hours. FIG. 4 illustrates a 3D scaffold printed, using the apparatus of FIGS. 3-3D, showing a top view of the EtOH75 (HA scaffold and the THF75_HA scaffold. The small bar in the bottom right corner of each image of FIG. 4 represents 5 mm. In the above-described examples, the inventors carried out the printing in a 0.1M sodium phosphate dibasic (Na₂HPO₄) solution that acted as a hardening accelerator. However, it will be understood that other hardening accelerators can also be used, and that different molarities for the solution can be used. For phosphate based hardening accelerators, molarities between about 0.01M and about 2.0M may be used. Within this range, at decreasing molarity, the hardening time can increase, which may require slower printing speeds to be used, whereas increasing molarity reduces the hardening time and allows higher printing speeds. If the molarity increases further, the hardening time may increase again. When the hardening accelerator is included in the slurry, the aqueous solution can also be water, and the accelerator dissolves in the water to provide an aqueous solution of the hardener.

After a scaffold structure is printed, it optionally may be submerged in an aqueous solution, including a hardening accelerator solution such as those described above. The structure may be submerged for up to an hour, or at least one hour, such as up to 10 hours, or up to 24 hours, or up to 48 hours or longer as may be desired. Submerging in the aqueous solution may further promote hardening and/or ensure removal of any residual solvents from the scaffold.

Characterization of the 3D Printed Scaffolds, Testing and Results In Situ HA Formation in 3D Printed Scaffolds X-Ray Diffraction (XRD) Analysis

Qualitative and quantitative information of the formation of HA in the scaffolds were obtained through X-ray diffraction (XRD) to reveal detailed information about chemical composition, crystallography, and structure of the scaffolds. XRD θ-2θ scans were collected on the PVB/HA scaffolds with dimensions of 17(L)×7(W)×1(H) mm at room temperature using a Philips Norelco diffractometer (vertical goniometer with automated scanning hardware) with Cu K-alpha radiation. The scanning range was from 10° to 60° with 0.03° 20 steps and a 3 second count time at each step (FIG. 5 ).

The XRD data of FIG. 5 suggest that the presence of the Na₂HPO₄ bath during the 3D-printing is necessary or at least advantageous for in situ HA formation. The XRD diffraction data for the printed scaffolds, as shown in FIG. 5 validates that hypothesis. The data of FIG. 5 show the XRD spectrum of the PVB/CPC composite scaffold printed in air (curve a), printed in the presence of water (DW) for 24 hours (curve b) and 48 hours (curve c), and printed in the presence of Na₂HPO₄ (SP) for 24 hours (curves d and f) and 48 hours (curves e and g). Specifically, the EtOH75 slurry initially was printed in the air (without the aqueous solution), resulting in a scaffold containing only TTCP and DCPA (20) as shown by curve a in FIG. 5 . Next, the EtOH75 slurry was printed in presence of a water bath, resulting in the absence of HA formation independently of the immersion time (24 hours or 48 hours—see curves b and c of FIG. 5 ). Finally, in the presence of a Na₂HPO₄ aqueous solution during either EtOH75 or THF75 3-D printing process, HA formation was detected by XRD. The test results show that both EtOH75_HA and THF75_HA, when embedded for 48 hours in the Na₂HPO₄ solution, provided proper HA formation with no unreactive TTCP and DCPA (see FIG. 5 , curves d, e, f, and g).

Scanning Electron Microscope Study

The detailed morphologies of the EtOH75_HA and THF75_HA were obtained by scanning electron microscope (SEM; JEOL, JSM-IT1500, MA) at an accelerating voltage of 10 kV. The SEM samples were vertically cut, mounted on aluminum sample studs, and coated with gold in the argon environment using a thin film sputter (Denton Vacuum, Desk V, NJ).

FIG. 6A provides a structural comparison of the two scaffolds, EtOH75_HA and THF75_HA. Scanning electron microscope (SEM) images from the “TOP” view (where the bar represents 500 μm), “CROSS-SECTIONAL” view (bar represents 500 μm) and “SINGLE FILAMENT” view (bar represents 100 μm) of the dried EtOH75_HA scaffold show different structures from those of the dried THF75_HA scaffold. As can be seen in the view marked “TOP,” the EtOH75_HA printed filaments are wider and the gaps between filaments are narrower than those of the THF75_HA scaffold. As can be seen in FIGS. 6A and 6B, SEM images from the top view and the cross-sectional view of the dried EtOH75_HA scaffolds show geometrical structures that differ from those of the THF75_HA scaffolds. Furthermore, the dimension (diameter) of EtOH75_HA printed filament (238.26±6.39 μm) was larger than the THF75_HA printed filament, which has a circular shape with dimension (164.98±6.85 μm) (the bar in FIG. 6B represents 10 μm).

Porosity of the Printed PVB/HA Composite Scaffolds

To further evaluate our porosity studies, the inventors performed micro-computed tomography (micro-CT) for the two scaffolds. The porosity of the PVB/HA scaffolds was imaged by using micro-computed tomography (micro-CT) (Scanco Medical, μCT 40, PA). The specimens (17(L)×5(W)×3(H) mm) were placed on the PMMA sample holder (U40830) between the X-ray source and the CCD camera, such that the whole specimen was encompassed in the field of view. The exposure conditions were 180° rotations, 45 kVp, and 177 μA. The porosity from the scanned images was calculated by μCT evaluation program V6.5 with the range of the threshold values (Min. 352, and Max. 1000). FIGS. 7A and 7B present representative views of the two scaffold structures at micro-CT. FIG. 7C presents comparative porosity values for the two scaffolds. The THF75_HA scaffold showed higher porosity (60.60%) compared to the EtOH75_HA scaffold (43.8%).

Surface Area

FIG. 7D illustrates the disparity in surface area between the two scaffolds, with the EtOH75_HA showing an approximately 33% larger surface area than the THF75_HA scaffold.

Mechanical Properties

Scaffold geometry and microstructure are related to the mechanical properties of the scaffolds. To expand the herein disclosed studies and to further characterize the mechanical properties of the 3D printed scaffolds, the inventors performed tensile strength and compressive strength tests, the results of which are shown, respectively, in FIGS. 8A-D, and 9A-9D. Ultimate Tensile Strength (UTS) Measurement

The mechanical properties of EtOH75 and THF75 scaffolds with the dimension of 17(L)×7(W)×1(H) mm were measured using the universal tensile machine (Instru-Met Cop., Model 1122, NJ) with 1 kN load cell. The scaffolds were vertically mounted on two sample holders with adhesion glue. Load-deformation data were recorded at a crosshead speed of 1 mm/min, and then, Young's modulus, yield strength, and the ultimate tensile strength were calculated through the stress-strain curve.

For the tensile strength test, the stress-strain curve for both scaffolds showed the initial elastic, plastic deformation, and rapid increase in stress (FIG. 8A). Although Young's modulus and yield strength of EtOH75_HA scaffolds were similar to the THF75_HA scaffold, the ultimate tensile strength (UTS) in EtOH75_HA scaffolds (3.72±0.97 MPa) was higher by 2-fold compared to THF75_HA scaffold (2.0±0.14 MPa) (FIGS. 8B, C, D).

Compressive Strength Measurement

The compressive strength tests show results similar to the tensile strength tests. See FIG. 9A. The compressive modulus and compressive yield strength of EtOH75_HA and THF75_HA show similar values, but ultimate compressive strength in EtOH75_HA scaffolds (7.74±1.13 MPa) was higher by 3.4-fold than that of THF75_HA scaffold (2.28±1.22 MPa) (FIGS. 9B, C, D). The porosity tests and strength measurements, taken together, illustrate that the microstructure of the THF75_HA scaffold has a higher porosity and a more brittle behavior than the EtOH75 HA scaffold.

Osteoconductivity of the 3D Printed Scaffolds

The scaffolds were assayed for osteogenesis by staining for Alkaline Phosphatase with Leukocyte Alkaline Phosphatase kit (Sigma), for protein-associated calcification with Alizarin

Red S (AR), and for mineral deposition with Silver Nitrate solution (Von Kossa: VK). The scaffolds were fixed in 4% paraformaldehyde (PFA; Sigma) for 20 minutes at 37° C., washed twice in PBS, permeabilized with 0.1% (v/v) Triton X 100 in PBS for 20 minutes at RT, and treated with blocking solution (0.01% (v/v) Triton X 100, 5% (w/v) goat serum (Sigma) in PBS) overnight at 4° C. Next day, DAPI (1:1000, Sigma), and Alexa Fluor 647 Phalloidin (1:200, Thermo Fisher) were added and incubated overnight at 4° C. Finally, the cells were stained for cell viability/cytotoxicity by using LIVE/DEAD™ Viability/Cytotoxicity Kit (ThermoFisher). The scaffolds were imaged using a confocal microscope (LSM 800, Carl Zeiss), and image analysis made by Image J by performing a maximum intensity z projection and merging the channels.

After the scaffold characterization, cytocompatibility and osteoconductivity of the 3D printed scaffolds were tested. Initially, human osteoblasts (HOBS) attached and spread on the 3D printed scaffolds. To test the mineralization and osteoconductivity potential of the cells on the 3D printed scaffolds, Alkaline Phosphate (ALP), Alizarin Red (AR), and Von Kossa (VK) staining were performed. The cells cultured on the EtOH75_HA scaffold under mineralization conditions showed higher mineralization (˜2-fold) compared to the THF75_HA scaffold as shown in FIGS. 10A-10C.

Cell Culture

In examples, the culture media conditions for the cells can be selected from a group consisting of β-glycerophosphate, ascorbic acid, serum, Vitamin D, Vascular growth factor (VEGF), platelet growth factor (PDGF), Prostaglandin E2 (PGE2), Bone morphogenic protein-2 (BMP-2), Bone morphogenic protein-6 (BMP-6), Bone morphogenic protein-7 (BMP-7), connective growth factor (CTGF), basic fibroblast growth factor (bFGF), receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), transforming growth factor beta (TGFbeta), insulin, sphingosine-1 phosphate (S1P), Phorbol 12-myristate 13-acetate (PMA), Bovine serum albumin (BSA), hydrocortisone, cortisol, EGF (Epidermal growth factor), HGF (Hepatocyte Growth Factor), Osteoblast Growth Medium Supplement Mix (OGM), Osteoblast Mineralization Medium (OMM), and combinations thereof.

In an example, primary HOBS (Promocell) were cultured in Osteoblast Growth Medium Supplement Mix (OGM) (Promocell). All experiments were performed with HOBS at passage 4 to 5. Finally, for differentiation assays, the HOBS were plated on 3D printed scaffolds (3(L)×3(W)×0.5(H) mm) at 0.5 million/ml density. All scaffolds samples were sterilized with 70% Ethanol and treated ultraviolet (UV) irradiation for 12 hours. The next day, the cells were exposed to Osteoblast Mineralization Medium (OMNI) (Promocell) for 10 days in a humidified incubator in an atmosphere containing 5% v/v CO₂ at 37° C.

Initially, the inventors confirmed the OBs attachment and spread on the 3D printed scaffold by F-actin (phalloidin) staining (FIG. 13 ). The cells were spread on the printed filaments as indicated in FIG. 13 . The OBs were stained for F-actin (phalloidin) and DAPI for the nuclei (OB nuclei). After 14 days OGM culturing the cells, their viability was tested on the Live/Dead assay (FIGS. 14A and 14B). Next, the inventors tested the mineralization and osteoconductivity potential of the cells on the 3D printed scaffolds by Alkaline Phosphate (ALP), Alzarin Red (AR), and Von Kossa (VK) staining. OBs on EtOH75_HA and THF75_HA scaffolds were cultured in OMNI conditions for 14 days demonstrated a 1.5-fold increase compared to OGM conditions based on ALP (FIG. 10A). Similarly, cells cultured under OMM conditions in both scaffolds showed 1.5-fold increase based on AR staining (FIG. 10B). Finally, our VK study showed that the cells cultured in OMNI conditions exhibited 2-fold and 1.5-fold increase in EtOH75_HA and THF75_HA, respectively. Additionally, the cells cultured on the EtOH75_HA scaffold showed higher mineralization capacity (˜2-fold) compared to the THF75_HA scaffold (FIG. 10C). To further evaluate the molecular mechanisms associated with the bone regeneration activity of OBs on EtOH75_HA and THF75_HA scaffolds, we analyzed their capacity to regulate the genes involved in osteoblast differentiation and mineralization. The expression of the typical OB-specific genes, including runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), integrin (33 (ITGB3), and collagen type I alpha 2 (COL1A2), was determined. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the expression of RunX2, ALP, OPN, OCN, ITGB3, and COL1A2 increased in OMM cultures compared to those in OGM cultures (FIGS. 11A-11F). These results clearly demonstrated that OB differentiation gene expression was dramatically up-regulated in OMNI cultures of OB seeded scaffolds.

Statistical Analysis

Statistical analysis of the quantitative data was conducted by one-way Analysis of Variance (ANOVA) using SPSS software. The p-values of less than 0.05 were considered as significant.

Biocompatibility

The 3D-printed scaffolds demonstrated osteoconductivity based on the ALP, AR and VK staining and relative mRNA level expression of key osteogenic markers such as COL1A2, ALP. The 3D-printed scaffolds may be used to support growth of different cell types or combination of cell types, including:

-   -   Mesenchymal stem cells (MSCs), which are differentiating to         osteoblasts;     -   Induced pluripotent stem cells (IPSCs), which are         differentiating to osteoblasts;     -   Osteoblasts;     -   Pre-osteoblasts;     -   Osteoids;     -   Osteoclasts precursor cells;     -   Peripheral blood mononuclear cells (PBMC), which are         differentiating to osteoclasts;     -   Endothelial progenitor cells (EPC);     -   Endothelial cells;     -   Macrophages; and     -   Neutrophils.

Additionally, these cells may be encapsulated as necessary and then mixed with CPC/polymer in the slurry (thereby forming a bio ink), or they may be included in a bio ink provided in a second syringe extruder. The advantage is that the cell-containing slurries or inks may be printed on specific locations in the CPC-scaffolds, thereby offering direct integration of the cells into the scaffold, avoiding a 14-day culture on the scaffolds, and faster osteointegration and repair with the native tissue.

Enumerated Embodiments

In some embodiments, the present invention is directed to the following non-limiting embodiments:

Embodiment 1 provides a three-dimensional, biocompatible precursor composition for room-temperature printing a three-dimensional (3D) bio-compatible polymer/bone cement composite scaffold, the precursor composition comprising:

-   -   a room-temperature slurry comprising a mixture of:         -   a solid phase comprising at least one calcium-containing             compound selected from a first group consisting of             tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous             (DCPA), α-tricalcium phosphate (α-TCP), β-tricalcium             phosphate (β-TCP), amorphous calcium phosphate (ACP),             octacalcium phosphate (OCP), dicalcium phosphate dihydrate             (DCPD), monocalcium phosphate monohydrate (MCPM),             monocalcium phosphate anhydrous (MCPA), calcium sulfate             (CaSO₄), calcium sulfate hemihydrate (α- or β-CaSO₄.0.5H₂O),             calcium carbonate (CaCO₃), calcium sulfate dihydrate             (CaSO₄.2H₂O), calcium oxide (CaO), and calcium hydroxide             (Ca(OH)₂), and         -   a liquid phase comprising a polymer in at least one             non-aqueous solvent, wherein the polymer is insoluble or has             low solubility in water,             wherein a weight ratio of the solid phase to the liquid             phase is between 0.1 to 1 and 3 to 1.

Embodiment 2 provides the precursor composition of Embodiment 1, wherein the weight ratio of the solid phase to the liquid phase is between 0.75 to 1 and 3 to 1.

Embodiment 3 provides the precursor composition of Embodiment 1, wherein the at least one calcium-containing compound in the solid phase comprises at least one of tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and dicalcuim phosphate anhydrous (DCPA; CaHPO₄).

Embodiment 4 provides the precursor composition of Embodiment 3, wherein the TTCP is provided with a particle size in a range from 1 to 17 μm and wherein the DCPA is provided with a particle size in a range from 1 to 5 μm.

Embodiment 5 provides the precursor composition of Embodiment 3, wherein the TTCP and DCPA are provided with a weight ratio TTCP:DCPA in a range of about 97%:3% to 8%:92%.

Embodiment 6 provides the precursor composition of Embodiment 3, wherein the solid phase compounds have a Ca/P molar ratio between 1 and 2.

Embodiment 7 provides the precursor composition of Embodiment 1, wherein the polymer is selected from a polymer group consisting of polyvinyl butyral (PVB), polycaprolactone (PCL), poly lactic-co-glycolic acid (PLGA), Poly(ethylene glycol), Polyvinyl pyrrolidone (PVP), Poly(methyl methacrylate) (PMMA), Polyoxazoline, polyphosphoesters (PPE), Poly-L-latic acid (PLLA), Polyacrylic acid (PAA), and Dextran.

Embodiment 8 provides the precursor composition of Embodiment 1, wherein the at least one non-aqueous solvent is selected from a solvent group consisting of ethanol (EtOH), tetrahydrofuran (THF), acetic acid, acetone, methanol, 2-propanol, butanol, 2-butoxyethanol, cyclohexanone, benzyl alcohol, 1-methoxy-propanol-2, butyl glycol, n-butyl, acetate, ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylsulfoxide, NMP, chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, cyclohexanone and 2-nitropropane, acetone, 2-butanone, ethyl acetate, dimethylformamide, acetonitrile, dichloromethane, chloroform, and ethyl acetate.

Embodiment 9 provides the precursor composition of Embodiment 1, further comprising a hardening accelerator, wherein the hardening accelerator is selected from a second group consisting of sodium phosphate dibasic, monosodium phosphate, trisodium phosphate, ammonium phosphate, ammonium dihydrogen phosphate, monopotassium phosphate, dipotassium phosphate, tripotassium phosphate, sodium fluoride, potassium fluoride, sodium acetate, potassium oxalate, sodium sulfate, sodium cacodylate, and organic acid, and wherein the organic acid is selected from a third group consisting of glycolic acid, citric acid, tartaric acid, malonic acid, malic acid, and maleic acid.

Embodiment 10 provides the precursor composition of Embodiment 9, wherein the hardening accelerator makes up to 10% of the solid phase by weight.

Embodiment 11 provides the precursor composition of Embodiment 9, wherein the scaffold is printed submerged in an aqueous bath.

Embodiment 12 provides the precursor composition of Embodiment 1, wherein the scaffold is printed submerged in an aqueous bath and wherein a hardening accelerator is added to the aqueous bath.

Embodiment 13 provides the precursor composition of Embodiment 1, wherein the solid phase further comprises a compound selected from a fourth group consisting of a carbonate, chloride (Cl), an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, a quaternary ammonium fluoride, a fluorosilicate, and a monofluorophosphate.

Embodiment 14 provides the precursor composition of Embodiment 1, wherein the slurry further comprises a bioactive material selected from a fifth group consisting of osteoblasts, osteoclasts, osteids, endothelial cells, endothelial progenitor cells, neutrophils, macrophages, and combinations thereof.

Embodiment 15 provides a precursor composition that, when printed at room temperature, forms a 3D bio-compatible polymer/calcium phosphate cement composite scaffold, the precursor composition comprising:

-   -   a room temperature slurry comprising:         -   a solid phase comprising a mixture of one or more calcium             phosphate compounds selected from a first group consisting             of TTCP, α-TCP and β-TCP, ACP, OCP, DCPA, DCPD, MCPM, and             MCPA, and         -   a liquid phase comprising a polymer in a solvent, the             solvent being selected from a first solvent group consisting             of ethanol (EtOH) and tetrahydrofuran (THF); and     -   a hardening accelerator, which is added during printing of the         polymer/calcium phosphate cement composite scaffold.

Embodiment 16 provides the precursor composition of Embodiment 15, wherein the hardening accelerator is selected from a second group consisting of sodium phosphate dibasic (Na₂HPO₄), ammonium phosphate ((NH₄)₃PO₄), potassium phosphate dibasic (K₂HPO₄), sodium fluoride (NaF), potassium fluoride (KF), sodium acetate, potassium oxalate (C₂K₂O₄), sodium sulfate (Na₂SO₄), and sodium cacodylate.

Embodiment 17 provides the precursor composition of Embodiment 15, wherein the precursor composition is printed in an aqueous bath.

Embodiment 18 provides the precursor composition of Embodiment 17, wherein the hardening accelerator is added to the aqueous bath during scaffold printing.

Embodiment 19 provides the precursor composition of Embodiment 15,

-   -   wherein the polymer is selected from a first polymer group         consisting of polyvinyl butyral (PVB) and polycaprolactone         (PCL);     -   wherein the PVB is dissolved in a solvent selected from a second         solvent group consisting of acetic acid, acetone, methanol,         2-propanol, butanol, 2-butoxyethanol, cyclohexanone, benzyl         alcohol, 1-methoxy-propanol-2, butyl glycol, n-butyl, acetate,         ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide,         N,N-dimethylsulfoxide, and NMP; and     -   wherein the PCL is dissolved in a solvent selected from a third         solvent group consisting of chloroform, dichloromethane, carbon         tetrachloride, benzene, toluene, cyclohexanone, 2-nitropropane,         acetone, 2-butanone, ethyl acetate, dimethylformamide, and         acetonitrile.

Embodiment 20 provides the precursor composition of Embodiment 15, wherein the solid phase compounds comprise TTCP and DCPA and have a Ca/P molar ratio in a range from 1.33 to 1.90.

Embodiment 21 provides the precursor composition of Embodiment 15, further comprising a compound selected from a fourth group consisting of a carbonate, chloride (Cl), an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, a quaternary ammonium fluoride, a fluorosilicate, and a monofluorophosphate.

Embodiment 22 provides a computer-controlled method for room temperature 3D printing a biocompatible, composition-controlled scaffold, the method comprising:

-   -   preparing a solid phase composition comprising a         calcium-containing cement powder;     -   preparing a liquid phase composition comprising a polymer         material dissolved in a solvent;     -   homogeneously mixing the solid phase composition and the liquid         phase composition to create a homogeneous, bio-compatible         slurry;     -   disposing the slurry in a reservoir system coupled to a printing         nozzle system, the printing nozzle system comprising at least         one printing nozzle;     -   submerging a printing substrate in a liquid bath disposed below         the printing nozzle;     -   under control of a computer, operating a motor to extrude the         slurry, at room temperature, from the reservoir system through         the printing nozzle system and to cause relative x, y, and z         displacement between the printing nozzle system and the printing         substrate;     -   employing a hardening accelerator to assist formation of the         biocompatible, composition-controlled scaffold; and     -   maintaining the 3D printing scaffold fully submerged in the         liquid bath during an entire 3D printing process.

Embodiment 23 provides the computer-controlled method of Embodiment 22, wherein the liquid bath is an aqueous solution containing the hardening accelerator, wherein the hardening accelerator is selected from a first group consisting of sodium phosphate dibasic (Na₂HPO₄), ammonium phosphate ((NH₄)₃PO₄), potassium phosphate dibasic (K₂HPO₄), sodium fluoride (NaF), potassium fluoride (KF), sodium acetate, potassium oxalate (C₂K₂O₄), sodium sulfate (Na₂SO₄), and sodium cacodylate.

Embodiment 24 provides the computer-controlled method of Embodiment 22, further comprising adding to the solid phase composition, a compound selected from a second group consisting of carbonate (NaHCO₃), chloride (Cl), and sodium fluoride (NaF).

Embodiment 25 provides the computer-controlled method of Embodiment 22,

-   -   wherein the polymer in the slurry is selected from a polymer         group consisting of: PVB, Polycaprolactone (PCL), Poly         Lactic-co-Glycolic acid (PLGA), Poly-L-lactic acid (PLLA),         Poly(ethylene glycol), Polyvinyl pyrrolidone (PVP), Poly(methyl         methacrylate) (PMMA), Polyoxazoline, polyphosphoesters (PPE),         and Dextran;     -   wherein the PVB is dissolved in a solvent selected from a first         solvent group consisting of acetic acid, acetone, methanol,         ethanol, 2-propanol, butanol, 2-butoxyethanol, cyclohexanone,         benzyl alcohol, 1-methoxy-propanol-2, butyl glycol, n-butyl,         acetate, ethyl acetate, N,N-dimethylacetamide,         N,N-dimethylformamide, N,N-dimethylsulfoxide, NMP, and THF;     -   wherein the PCL is dissolved in a in a solvent selected from a         second solvent group consisting of THF, chloroform,         dichloromethane, carbon tetrachloride, benzene, toluene,         cyclohexanone and 2-nitropropane, acetone, 2-butanone, ethyl         acetate, dimethylformamide, and acetonitrile;     -   wherein the PLGA is dissolved in a in a solvent selected from a         third solvent group consisting of THF, acetone, ethyl acetate,         and chlorinated solvents;     -   wherein the PLLA is dissolved in a solvent selected from a         fourth solvent group consisting of chloroform and         dichloromethane (DCM);     -   wherein the PLLA is dissolved in a solvent selected from a fifth         solvent group consisting of acetone, dichloromethane, ethanol         (95%), methanol, benzene, glycerin, and glycols;     -   wherein the PVP is dissolved in a solvent selected from a sixth         solvent group consisting of methanol and ethanol;     -   wherein the PMMA is dissolved in a solvent selected from a         seventh solvent group consisting of THF, methanol, and ethanol;     -   wherein the PMMA is dissolved in a solvent selected from an         eighth solvent group consisting of toluene, dichloromethane,         chloroform, and acetone;     -   wherein the PPE is dissolved in a solvent selected from a ninth         solvent group consisting of THF, acetonitrile, chloroform, ethyl         acetate, and Poly((lactide-co-ethylene         glycol)-co-ethyloxyphosphate)); and     -   wherein the Dextran is dissolved in a solvent selected from a         tenth solvent group consisting of methyl sulfide, formamide,         ethylene glycol, and glycerol.

Embodiment 26 provides the computer-controlled method of Embodiment 22, further comprising disposing the hardening accelerator in the reservoir system, the liquid bath, and both the reservoir system and the liquid bath.

Embodiment 27 provides the computer-controlled method of Embodiment 22, further comprising disposing in the reservoir system, the liquid bath, and both the reservoir system and the liquid bath, one or more compounds chosen from a third group consisting of carbonate

(NaHCO₃), sodium chloride (NaCl), an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, a quaternary ammonium fluoride, a fluorosilicate, and a monofluorophosphate.

Embodiment 28 provides the computer-controlled method of Embodiment 22, further comprising incorporating cells during printing, the cells being selected from a group consisting of osteoblasts, osteoclasts, osteoids, endothelial cells, endothelial progenitor cells, neutrophils, macrophages, and combinations thereof.

Embodiment 29 provides the computer-controlled method of Embodiment 22, further comprising incorporating cells into the printed scaffold, the cells being selected from a group consisting of osteoblasts, osteoclasts, osteoids, endothelial cells, endothelial progenitor cells, neutrophils, macrophages, and combinations thereof. 

What is claimed is:
 1. A three-dimensional, biocompatible precursor composition for room-temperature printing a three-dimensional (3D) bio-compatible polymer/bone cement composite scaffold, the precursor composition comprising: a room-temperature slurry comprising a mixture of: a solid phase comprising at least one calcium-containing compound selected from a first group consisting of tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), α-tricalcium phosphate (α-TCP), β-tricalcium phosphate (β-TCP), amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), dicalcium phosphate dihydrate (DCPD), monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), calcium sulfate (CaSO₄), calcium sulfate hemihydrate (α- or β-CaSO₄.0.5H₂O), calcium carbonate (CaCO₃), calcium sulfate dihydrate (CaSO₄.2H₂O), calcium oxide (CaO), and calcium hydroxide (Ca(OH)₂), and a liquid phase comprising a polymer in at least one non-aqueous solvent, wherein the polymer is insoluble or has low solubility in water, wherein a weight ratio of the solid phase to the liquid phase is between 0.1 to 1 and 3 to
 1. 2. The precursor composition of claim 1, wherein the weight ratio of the solid phase to the liquid phase is between 0.75 to 1 and 3 to
 1. 3. The precursor composition of claim 1, wherein the at least one calcium-containing compound in the solid phase comprises at least one of tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and dicalcuim phosphate anhydrous (DCPA; CaHPO₄).
 4. The precursor composition of claim 3, wherein the TTCP is provided with a particle size in a range from 1 to 17 μm and wherein the DCPA is provided with a particle size in a range from 1 to 5 μm.
 5. The precursor composition of claim 3, wherein the TTCP and DCPA are provided with a weight ratio TTCP:DCPA in a range of about 97%:3% to 8%:92%.
 6. The precursor composition of claim 3, wherein the solid phase compounds have a Ca/P molar ratio between 1 and
 2. 7. The precursor composition of claim 1, wherein the polymer is selected from a polymer group consisting of polyvinyl butyral (PVB), polycaprolactone (PCL), poly lactic-co-glycolic acid (PLGA), Poly(ethylene glycol), Polyvinyl pyrrolidone (PVP), Poly(methyl methacrylate) (PMMA), Polyoxazoline, polyphosphoesters (PPE), Poly-L-latic acid (PLLA), Polyacrylic acid (PAA), and Dextran.
 8. The precursor composition of claim 1, wherein the at least one non-aqueous solvent is selected from a solvent group consisting of ethanol (EtOH), tetrahydrofuran (THF), acetic acid, acetone, methanol, 2-propanol, butanol, 2-butoxyethanol, cyclohexanone, benzyl alcohol, 1-methoxy-propanol-2, butyl glycol, n-butyl, acetate, ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylsulfoxide, NMP, chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, cyclohexanone and 2-nitropropane, acetone, 2-butanone, ethyl acetate, dimethylformamide, acetonitrile, dichloromethane, chloroform, and ethyl acetate.
 9. The precursor composition of claim 1, further comprising a hardening accelerator, wherein the hardening accelerator is selected from a second group consisting of sodium phosphate dibasic, monosodium phosphate, trisodium phosphate, ammonium phosphate, ammonium dihydrogen phosphate, monopotassium phosphate, dipotassium phosphate, tripotassium phosphate, sodium fluoride, potassium fluoride, sodium acetate, potassium oxalate, sodium sulfate, sodium cacodylate, and organic acid, and wherein the organic acid is selected from a third group consisting of glycolic acid, citric acid, tartaric acid, malonic acid, malic acid, and maleic acid.
 10. The precursor composition of claim 9, wherein the hardening accelerator makes up to 10% of the solid phase by weight.
 11. The precursor composition of claim 9, wherein the scaffold is printed submerged in an aqueous bath.
 12. The precursor composition of claim 1, wherein the scaffold is printed submerged in an aqueous bath and wherein a hardening accelerator is added to the aqueous bath.
 13. The precursor composition of claim 1, wherein the solid phase further comprises a compound selected from a fourth group consisting of a carbonate, chloride (Cl), an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, a quaternary ammonium fluoride, a fluorosilicate, and a monofluorophosphate.
 14. The precursor composition of claim 1, wherein the slurry further comprises a bioactive material selected from a fifth group consisting of osteoblasts, osteoclasts, osteids, endothelial cells, endothelial progenitor cells, neutrophils, macrophages, and combinations thereof.
 15. A precursor composition that, when printed at room temperature, forms a 3D bio-compatible polymer/calcium phosphate cement composite scaffold, the precursor composition comprising: a room temperature slurry comprising: a solid phase comprising a mixture of one or more calcium phosphate compounds selected from a first group consisting of TTCP, α-TCP and β-TCP, ACP, OCP, DCPA, DCPD, MCPM, and MCPA, and a liquid phase comprising a polymer in a solvent, the solvent being selected from a first solvent group consisting of ethanol (EtOH) and tetrahydrofuran (THF); and a hardening accelerator, which is added during printing of the polymer/calcium phosphate cement composite scaffold.
 16. The precursor composition of claim 15, wherein the hardening accelerator is selected from a second group consisting of sodium phosphate dibasic (Na₂HPO₄), ammonium phosphate ((NH₄)₃PO₄), potassium phosphate dibasic (K₂HPO₄), sodium fluoride (NaF), potassium fluoride (KF), sodium acetate, potassium oxalate (C₂K₂O₄), sodium sulfate (Na₂SO₄), and sodium cacodylate.
 17. The precursor composition of claim 15, wherein the precursor composition is printed in an aqueous bath.
 18. The precursor composition of claim 17, wherein the hardening accelerator is added to the aqueous bath during scaffold printing.
 19. The precursor composition of claim 15, wherein the polymer is selected from a first polymer group consisting of polyvinyl butyral (PVB) and polycaprolactone (PCL); wherein the PVB is dissolved in a solvent selected from a second solvent group consisting of acetic acid, acetone, methanol, 2-propanol, butanol, 2-butoxyethanol, cyclohexanone, benzyl alcohol, 1-methoxy-propanol-2, butyl glycol, n-butyl, acetate, ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylsulfoxide, and NMP; and wherein the PCL is dissolved in a solvent selected from a third solvent group consisting of chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, cyclohexanone, 2-nitropropane, acetone, 2-butanone, ethyl acetate, dimethylformamide, and acetonitrile.
 20. The precursor composition of claim 15, wherein the solid phase compounds comprise TTCP and DCPA and have a Ca/P molar ratio in a range from 1.33 to 1.90.
 21. The precursor composition of claim 15, further comprising a compound selected from a fourth group consisting of a carbonate, chloride (Cl), an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, a quaternary ammonium fluoride, a fluorosilicate, and a monofluorophosphate.
 22. A computer-controlled method for room temperature 3D printing a biocompatible, composition-controlled scaffold, the method comprising: preparing a solid phase composition comprising a calcium-containing cement powder; preparing a liquid phase composition comprising a polymer material dissolved in a solvent; homogeneously mixing the solid phase composition and the liquid phase composition to create a homogeneous, bio-compatible slurry; disposing the slurry in a reservoir system coupled to a printing nozzle system, the printing nozzle system comprising at least one printing nozzle; submerging a printing substrate in a liquid bath disposed below the printing nozzle; under control of a computer, operating a motor to extrude the slurry, at room temperature, from the reservoir system through the printing nozzle system and to cause relative x, y, and z displacement between the printing nozzle system and the printing substrate; employing a hardening accelerator to assist formation of the biocompatible, composition-controlled scaffold; and maintaining the 3D printing scaffold fully submerged in the liquid bath during an entire 3D printing process.
 23. The computer-controlled method of claim 22, wherein the liquid bath is an aqueous solution containing the hardening accelerator, wherein the hardening accelerator is selected from a first group consisting of sodium phosphate dibasic (Na₂HPO₄), ammonium phosphate ((NH₄)₃PO₄), potassium phosphate dibasic (K₂HPO₄), sodium fluoride (NaF), potassium fluoride (KF), sodium acetate, potassium oxalate (C₂K₂O₄), sodium sulfate (Na₂SO₄), and sodium cacodylate.
 24. The computer-controlled method of claim 22, further comprising adding to the solid phase composition, a compound selected from a second group consisting of carbonate (NaHCO₃), chloride (Cl), and sodium fluoride (NaF).
 25. The computer-controlled method of claim 22, wherein the polymer in the slurry is selected from a polymer group consisting of: PVB, Polycaprolactone (PCL), Poly Lactic-co-Glycolic acid (PLGA), Poly-L-lactic acid (PLLA), Poly(ethylene glycol), Polyvinyl pyrrolidone (PVP), Poly(methyl methacrylate) (PMMA), Polyoxazoline, polyphosphoesters (PPE), and Dextran; wherein the PVB is dissolved in a solvent selected from a first solvent group consisting of acetic acid, acetone, methanol, ethanol, 2-propanol, butanol, 2-butoxyethanol, cyclohexanone, benzyl alcohol, 1-methoxy-propanol-2, butyl glycol, n-butyl, acetate, ethyl acetate, N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylsulfoxide, NMP, and THF; wherein the PCL is dissolved in a in a solvent selected from a second solvent group consisting of THF, chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, cyclohexanone and 2-nitropropane, acetone, 2-butanone, ethyl acetate, dimethylformamide, and acetonitrile; wherein the PLGA is dissolved in a in a solvent selected from a third solvent group consisting of THF, acetone, ethyl acetate, and chlorinated solvents; wherein the PLLA is dissolved in a solvent selected from a fourth solvent group consisting of chloroform and dichloromethane (DCM); wherein the PLLA is dissolved in a solvent selected from a fifth solvent group consisting of acetone, dichloromethane, ethanol (95%), methanol, benzene, glycerin, and glycols; wherein the PVP is dissolved in a solvent selected from a sixth solvent group consisting of methanol and ethanol; wherein the PMMA is dissolved in a solvent selected from a seventh solvent group consisting of THF, methanol, and ethanol; wherein the PMMA is dissolved in a solvent selected from an eighth solvent group consisting of toluene, dichloromethane, chloroform, and acetone; wherein the PPE is dissolved in a solvent selected from a ninth solvent group consisting of THF, acetonitrile, chloroform, ethyl acetate, and Poly((lactide-co-ethylene glycol)-co-ethyloxyphosphate)); and wherein the Dextran is dissolved in a solvent selected from a tenth solvent group consisting of methyl sulfide, formamide, ethylene glycol, and glycerol.
 26. The computer-controlled method of claim 22, further comprising disposing the hardening accelerator in the reservoir system, the liquid bath, and both the reservoir system and the liquid bath.
 27. The computer-controlled method of claim 22, further comprising disposing in the reservoir system, the liquid bath, and both the reservoir system and the liquid bath, one or more compounds chosen from a third group consisting of carbonate (NaHCO₃), sodium chloride (NaCl), an alkali metal fluoride, an alkaline earth metal fluoride, a silver-based fluoride, stannous fluoride, ammonium fluoride, a quaternary ammonium fluoride, a fluorosilicate, and a monofluorophosphate.
 28. The computer-controlled method of claim 22, further comprising incorporating cells during printing, the cells being selected from a group consisting of osteoblasts, osteoclasts, osteoids, endothelial cells, endothelial progenitor cells, neutrophils, macrophages, and combinations thereof.
 29. The computer-controlled method of claim 22, further comprising incorporating cells into the printed scaffold, the cells being selected from a group consisting of osteoblasts, osteoclasts, osteoids, endothelial cells, endothelial progenitor cells, neutrophils, macrophages, and combinations thereof. 